This project is a continuation of a broadly based investigation into the regulatory mechanisms that control the production of virulence factors and other exoproteins in Staphylococcus aureus, with an extension to the regulation of superantigen toxin synthesis in Streptococcus pyogenes. The present focus is on the mechanism of action of the agr effector molecule, the 514 nt RNAIII, and is based on the hypothesis that RNAIII interacts with internal regulatory mediators. We have found that a key intracellular mediator is the 2-component sae system, which is transcriptionally activated by RNAIII and which is required for the expression of many of the exoprotein genes. There are four specific aims: 1. To characterize the central pathway from agr via RNAIII to target genes. 2. To determine the mechanisms by which external stimuli and certain growth conditions affect the expression of genes in the agr regulon. 3. To analyze by means of reporter gene fusions the expression of different regulatory and target genes. 4. To determine whether the regulation of speA and other toxin genes by group A hemolytic streptococci is similar to staphylococcal regulation of virulence factors. In Aim 1, oligonucleotide arrays will be used to define the agr regulon and overlapping regulons governed by other global regulators of virulence. In Aim 2, the mechanism by which RNAIII activates the intracellular mediator sae will be identified, and its role in regulating target gene transcription defined. In Aim 3, the focus will be on the mechanism by which certain external stimuli interact with the agr pathway, starting with a determination of the sets of genes (stimulons) that are affected by particular environmental conditions. It is proposed to compare the effects of these stimuli and of various regulatory mutations in planktonic cultures with their effects in bioflims. In Aim 4, a continuation of a study in which we have identified an autoinducer of streptococcal erythogenic toxin (SPEA) synthesis, is proposed.